The overall purpose of this study is to understand how cells use actin- sequestering proteins to regulate levels of G- and F-actin and the organization of the cytoskeleton. The specific aims are, first, to investigate the structural basis for actin sequestration by thymosin Beta4 (Tbeta4) by identifying sites of contact through chemical crosslinking studies, by determining the packing of molecules in macrocrystals of the actin-TBeta4 complex, and by growing crystals suitable for structure determination by x-ray diffraction. The second aim is to determine how anchorage dependent cells such as fibroblast regulate the concentration of actin-sequestering proteins and G and F- actin levels in response to changes in substrate attachment and cell density; the third, related aim is to identify the actin-sequestering protein in fibroblasts. The fourth aim is to investigate the relationship between actin sequestering proteins and the changes in G- and F-actin levels in cells that undergo cytoskeletal reorganization in response to growth factors. This will be studied in PC12 cells which extend neurites when stimulated by nerve growth factor; some clones increase the transcription of Tbeta4 mRNA before and during neurite extension while others do not. We will use transfection with plasmids containing Tbeta4 cDNA (sense and antisense) to manipulate Tbeta4 levels in the presence and absence of growth factor stimulation and determine the effects on G-actin levels and on neurite extension. The fifth aim is to determine whether homologs of Tbeta4 are responsible for maintaining the large pools of sequestered actin in specific invertebrate cells of interest, using echinoderm eggs, an insect cell line, and the protozoan Dictyostelium discoideum. The regulation of G-actin pools and of cytoskeletal structure is intimately related to the regulation of cell attachment, growth, locomotion and cytokinesis. These processes are fundamental aspects of medically significant problems such as hemostasis, inflammation, the immune response and of normal and metastatic growth.